1. Field
The present application pertains generally to expression of genes in algae. In particular, the application relates to methods and compositions for introduction of genes and regulatory sequences into the nucleus of bioprocess algae.
2. Description of the Related Art
Culturing microalgae under nitrogen starvation promotes a shift in algae metabolism, decreasing the rates of protein synthesis and cell division. A concomitant increase in the total cellular content of lipid arises as the cell division rate decreases more rapidly than the rate of lipid synthesis (Livne and Sukenik, 1992). Subsequently, fatty acid profiles change in a species-specific manner, but the general trend observed is a reduction in polyenoic fatty acids and polar lipids, and an increase in nonpolar storage lipids (Zhila et al., 2005; Guschina and Harwood, 2006).
Pond systems for algae oil production may be able to increase total output of lipids and other useful biomass by the use of a biphasic system (Huntley and Redaljie, 2007) that shortens the duration of cultivation in open ponds. This reduces the undesired side effects of competition from contaminating species. Culture expansion can proceed under nonlimiting conditions, followed by a brief nitrogen starvation and enhanced synthesis of lipids. Made-to-order biosynthesis of lipids and other high value coproducts can be accomplished by induction or repression of fatty acid or other biosynthetic genes under the control of nitrogen-responsive promoters. Key lipid biosynthetic enzymes to regulate by this strategy include, but are not limited to, fatty acid synthases (Bonaventure et al., 2003; Wu et al., 1994), desaturases (Pidkowich et al., 2007) or the rate-limiting step for total output, accD (reviewed in Rawsthorne, 2002).
Until now, nitrogen-inducible or nitrate-responsive promoters have not been utilized in algae for this purpose. Ammonium (NH4++NH3) and nitrate (NO3−) are useful nitrogen sources for many microorganisms. High affinity ammonium transporters of the AMT/MEP family have been described at the molecular level in diverse organisms such as plants, yeast, bacteria, fungi and animals. The synthesis of structural and enzymatic genes required for nitrogen assimilation, such as NH3 gas channels (Gonzalez-Ballester et al., 2004; Kim et al., 2005) and nitrate reductase (Fernandez et al., 1989; Franco et al., 1984) is directly influenced in algae by environmental NH3 and NO3− conditions and is regulated primarily by controlling transcription. In Chlamydomonas, the ammonium transporter genes AMT1, AMT2 and AMT 4 are tightly repressed in the presence of ammonium or nitrate, and rapidly induced to a high level of transcription, three or four orders of magnitude in its absence (Kim et al., 2005). Nitrate reductase, NIT1, is similarly repressed by ammonium but it is further de-repressed in the presence of nitrate or in nitrogen-free medium (Fernandez et al., 1989).
Similarly, large-scale algae culture farms are ideal for sequestration of excess CO2 from point sources such as coal burning power plants. It is known that CO2 supplementation of cultures promotes high rates of alga photosynthesis and growth, and may also enhance lipid synthesis as much as 30% (Murandyan et al., 2004). It would be useful to use CO2 as an environmental signal for deliberate regulation of gene expression. However, until now, no CO2-inducible promoters have been identified and utilized in algae for this purpose. Regulatory sequences from a natural CO2 transporter system, such as Rh1, could provide a novel transgene expression system in algae that are responsive to varying algae culture conditions.
Other inducible promoter sequences useful for nuclear expression of genes for biofuels and process co-products include those responsive to light. For example, chlorophyll-binding proteins have been isolated, sequenced, and shown useful in diatoms for transgene expression.
Constitutive promoters are of interest in addition to inducible promoters for nuclear-based expression of genes. Constitutive viral promoter sequences have a long history of utility in biotechnology, effectively driving expression of transgenes in many in vivo culture platforms. In higher plants, pararetroviruses, such as Caulimovirus (CaMV) have proven especially useful. Although the host range of CaMV is restricted, its 35S rDNA promoter (Fang et al., 1989; Benfey et al., 1990) functions in many species of land plants and is used in most genetically modified crops (hypertext transfer protocol usbiotechreg.nbii.gov/). It has been used in the chlorophyte algae Chlamydomonas (Tang et al., 1995) and Dunaliella (Geng et al., 2003). This viral sequence was reported to work in the dinoflagellates Amphidinium and Symbodinium (Lohuis and Miller, 1998). However, it was not active when tested in the diatoms Cyclotella or Navicula (Dunahay et al., 1995). There is a clear need to discover promoter sequences from aquatic viruses, such as from the double-stranded DNA viruses in the Phycodnaviridae, as suitable substitutes for reliable and effective transgene expression in algae hosts. The Chlorella PBCV-1 virus produces more than 100 different proteins. The most abundant of these is the 54-kDa major coat protein, comprising more than 40 percent of the total mass of the virus. As such, it represents the most highly expressed protein gene and is an excellent candidate for driving constitutive expression of transgenes in Chlorophyte and other algae.
The ribosomal RNA (rRNA) genes of eukaryotes provide a remarkable example of gene duplication (Cortadas and Pavon, 1982). They may represent as much as 8% of the nuclear genome, as in Arabidopsis, for which approximately 570 polycistronic rDNA copies per haploid genome are clustered in two loci on separate chromosomes (Pruitt and Meyerowitz, 1986). The rRNA genes of eukaryotes are organized into a common structure encoding an RNA precursor that is processed into the mature 18s, 5.8S, and 28S rRNAs. Between the three coding regions are internal transcribed spacer regions (ITS-1 and ITS-2), and flanking the operon are the 5′ and 3′ external transcribed spacer regions (ETS). The operons are then supraorganized into tandem repeating units, separated by non-transcribed DNA known as the intergenic spacer (IGS) (FIG. 1A). IGS's are proposed to reduce silencing of nuclear transgenes due to effects on chromatin structure.